Efficiency of Triple Antibiotic Mixture and Propolis as Intracanal Medication in Revascularization process in immature apex: A clinical study

In the last 10 years, numerous published cases and case series described the revascularization or regenerative endodontics. Revascularization is a conservative method for inducing maturogenesis in necrotic immature teeth [1]. The favorable outcomes of regenerative endodontics are largely dependent on the adequate disinfection of the root canal [1]. These canals with compromised fragile underdeveloped dentinal walls represent a contraindication for mechanical instrumentation; thus, chemical debridement remains the main form of disinfection [1]. A mixture of ciprofl oxacin, metronidazole, and minocycline, known as the triple antibiotic paste (TAP), has been shown to be very effective in eliminating endodontic pathogens in vitro and in situ [1,3-5]. However, TAP has an adverse effect on stem cell survival [6]. Discoloration of the tooth is a problem mostly related to the use of minocycline in TAP [7]. Moreover, TAP can demineralize dentin resulting in reduced microhardness and fracture resistance [8]. Propolis, a fl avonoid-rich resinous product of honeybees, is ten times less cytotoxic than calcium hydroxide and has a well-known antibacterial effect [9]. Therefore, creating alternatives to the TAP for disinfecting the root canal of necrotic teeth during the process of pulp revascularization is thought to be of value. The null hypothesis in the current study stated that there is no difference between the tested intracanal medications on the outcome of the revascularization process in immature teeth.


Introduction
In the last 10 years, numerous published cases and case series described the revascularization or regenerative endodontics. Revascularization is a conservative method for inducing maturogenesis in necrotic immature teeth [1]. The favorable outcomes of regenerative endodontics are largely dependent on the adequate disinfection of the root canal [1]. These canals with compromised fragile underdeveloped dentinal walls represent a contraindication for mechanical instrumentation; thus, chemical debridement remains the main form of disinfection [1]. A mixture of ciprofl oxacin, metronidazole, and minocycline, known as the triple antibiotic paste (TAP), has been shown to be very effective in eliminating endodontic pathogens in vitro and in situ [1,[3][4][5]. However, TAP has an adverse effect on stem cell survival [6]. Discoloration of the tooth is a problem mostly related to the use of minocycline in TAP [7]. Moreover, TAP can demineralize dentin resulting in reduced microhardness and fracture resistance [8]. Propolis, a fl avonoid-rich resinous product of honeybees, is ten times less cytotoxic than calcium hydroxide and has a well-known antibacterial effect [9]. Therefore, creating alternatives to the TAP for disinfecting the root canal of necrotic teeth during the process of pulp revascularization is thought to be of value.
The null hypothesis in the current study stated that there is no difference between the tested intracanal medications on the outcome of the revascularization process in immature teeth.

Materials and Methods
Forty patients with immature, non-vital maxillary incisors were included in this study from the outpatient clinic of the Faculty of Dentistry, Ain Shams University, Cairo, Egypt. A detailed medical and dental history was obtained from each patient's parents or guardians. Only medically free patients were included in this research. The clinical and radiographic exclusion criteria were teeth with vertical fractures, periodontally involved teeth, and nonrestorable teeth. All procedures were performed after obtaining proper institutional review board approval based on the regulations of the Ethical Committee of the Faculty of Dentistry, Ain Shams University (FDASU-RECID011508). Intraoral periapical radiographs revealed immature apices. The age of the patients ranged between 8 and 18 years. Informed consent was signed for 2). Ciprofl oxacin + Propolis Paste: Ethanol extract of raw propolis (EEP; ElEzaby Co. Labs, Cairo, Egypt.) was prepared by adding 10 gm of propolis (Imtinan, Cairo, Egypt) to 40 gm of 70% ethanol (ElGomhorya Co., Cairo, Egypt) (for 20% tincture) in a dark container to prevent reduction of propolis. The container was sealed and placed at room temperature for a period of three weeks. The sealed container was manually shaken every 2 days to ensure proper mixing. After 3 weeks, the container was opened and ethanol extract of propolis was obtained. Ethanol-free EEP was made by evaporating the ethanol in a water bath [11,12]. EEP was then mixed with Ciprofl oxacin powder in the ratio 1:1. Saline drops were added and mixed using the pestle until a creamy paste was achieved.
3) Ciprofl oxacin + Metronidazole Paste: Ciprofl oxacin powder was mixed with Metronidazole powder in the ratio 1:1. Saline drops were added and mixed using the pestle until a creamy paste was achieved.

4) Propolis + Metronidazole paste: EEP was mixed with
Metronidazole powder in the ratio 1:1. Saline drops were added and mixed using the pestle until a creamy paste was achieved.
A preoperative radiograph was taken using the standardized paralleling technique by the Rinn XCP alignment system (Rinn Corporation Elgin, Illinois, USA) and Fona ScaNeo intra-oral digital imaging system (FONA Dental, Bratislava, Slovak Republic). Patients were treated as follows: Caries was excavated; access cavity was prepared. The rubber dam was then applied and working length was determined with a periapical radiograph taken with a fi le inserted into the canal. Canal space was debrided using K fi le size #80. Canal space was irrigated using 40 cc of NaOCl 2.6% solution and fi nal fl ush of saline. Canal space was dried using paper points. The antibiotic paste was prepared as previously described. One cubic centimeter of the prepared paste was injected into the canals using a sterile plastic syringe with 20" gauge needle.
Care was taken to avoid apical extrusion and to minimize placement in the coronal portion of the tooth. The access cavity was then sealed using temporary restoration (Coltosol F; Coltene Whaledent, Altstatten, Switzerland) over plain cotton. Coltosol F is a long-term temporary restoration. After the 3 weeks, under the same aseptic conditions, anesthesia without vasoconstrictor (Mepecaine, Alexandria Co., Alexandria, Egypt) was administrated. The tooth was re-entered, the antibiotic paste was removed and the canal was irrigated using a sterile saline and dried using paper points [4,13].
Sterile hand fi le size #25 was introduced into the root canal and placed at 2 mm beyond the working length to induce bleeding into the canal. The bleeding was allowed to reach a 3-mm level below the cemento-enamel junction, and teeth were left at rest for 5 minutes so that a blood clot could be formed [14]. Next, a 3-mm plug of MTA (Angelus; Londrina, Brazil) was inserted into the canals using a suitable-sized amalgam carrier to seal the root canal at the cervical level. The MTA plug was verifi ed radiographically. The MTA plug was then covered by moist cotton and temporary fi lling. After one week, MTA setting was confi rmed clinically, adhesive composite resin (Z250 Restorative; 3M ESPE, St Paul, Minnesota, USA) was used to seal the access cavity [15][16][17] Figure 1.

Evaluation:
Patients were recalled for follow up at 3, 6, 9, 12, and 18 months. Follow-up included the clinical assessment of pain and/or swelling, and standardized radiographic assessment for: 1. An increase in root length

A change in periapical bone density
All follow-up radiographs were standardized using the Rinn XCP alignment system and Fona ScaNeo intra-oral digital imaging system. All radiographic measures were collected by the same investigator. All radiographic measurements were repeated after 1 week, and the mean of the 2 sets was considered as the fi nal value.

Increase in root length:
A measuring scale was set in the Image-J software (Image-J v1.44, US National Institutes of Health, Bethesda, MD) by measuring a known clinical dimension to its radiographic dimension. The scale was calculated as number of measured pixels per mm length. Root length was measured as a straight line from the cementoenamel junction to the radiographic apex of the tooth [4,18] in millimeters. Pre and follow-up root lengths were measured using Image-J analysis software. Difference in length was calculated. Percentage of increase in length was calculated as follows: percentage of increase in length = [(postoperative length -preoperative length) / preoperative length] x 100.

Increase in root thickness:
Using the preset measurement scale, the level of the apical third was determined and fi xed from the cemento-enamel junction. The root thickness and the pulp width were measured mesiodistally at this level in millimeters. Mesiodistal dentin thickness was measured by subtraction of the pulp space from the whole root thickness. Pre and followup root thicknesses were measured. Measurements were done pre and post operatively at the same fi xed level [18]. Difference in dentin thickness was calculated. Percentage of increase in dentin thickness was calculated as follows: percentage of increase in dentin thickness = [(postoperative thicknesspreoperative thickness) / preoperative thickness] x 100.
Decrease in apical diameter: Using the preset measurement scale, the mesiodistal diameter of the apical foramen was measured in millimeters [4]. Measurements were done pre and postoperatively. Percentage of apical closure was calculated as follows: percentage of apical closure = [(preoperative apical diameter -postoperative apical diameter) / preoperative apical diameter] x 100.
Periapical bone density: Periapical bone density was estimated using Image-J software as follows: periapical area was located and analyzed for bone density. Average area density was measured in scale from 0 (black) to 255 (white) and recorded for each radiograph. The same area was then measured in subsequent radiographs and average densities were recorded for the follow up radiographs [4]. The difference between densities was calculated between subsequent radiographs [4,5]. Percentage of change in density was calculated from the original pre-operative radiograph density as follows: percentage of change in density = [(postoperative bone density -preoperative bone density)/preoperative bone density] x 100.

Result s
A total of fi ve cases were dropped out from the study, four of which was excluded due to poor patient compliance.
Clinical and radiographic examination during the follow-up period showed signs and symptoms of failure in 1 case, which was re-evaluated, and the treatment plan was shifted to MTA apexifi cation. Clinical outcomes are presented in Table 1.  increase in root length. However, no statistically signifi cant difference was found between 3 months and 6 months, nor between 6 months and 9 months, nor between 9 months and 12 months (P > 0.05) Table 2 Effect of time: Statistical analysis revealed signifi cant effect of time on increase in root length. However, no statistically signifi cant difference was found between 3 months and 6 months, nor between 6 months and 9 months, nor between 9 months and 12 months (P > 0.05) Table 2.
II. Increase in root thickness: Figure 3 Effect of type of intracanal medication: Statistical analysis showed that there was no statistically signifi cant difference between all groups through the whole follow up period (P > 0.05) Table 3.Effect of time: No statistically signifi cant difference was found between 6 months and 9 months (P > 0.05). However, statistically signifi cant difference was found among other follow up periods Table 3. III. Decrease in apical diameter: Figure 4 Effect of type of intracanal medication: Statistical analysis showed that there was no statistically signifi cant difference between all groups through the whole follow up period (P > 0.05) Table 4. Effect of time: Statistically signifi cant difference was found among all follow up periods Table 4.
IV. Periapical bone density: Figure 5 Effect of type of intracanal medication: Statistical analysis showed that there was no statistically signifi cant difference between all groups through the whole follow up period (P > 0.05) Table 5.

Effect of time:
Statistically signifi cant difference was found among all follow up periods. Signifi cant improvement in bone density was found after 9 months of follow up in each group Table 5.

Discussion
Triple antibiotic paste (TAP) has been found to have antimicrobial properties and to be biocompatible [19].
Ciprofl oxacin inhibits DNA gyrase synthesis, metronidazole inhibits DNA synthesis, and minocycline inhibits protein synthesis of microbes [15]. Because case reports have shown    The mean percentage of increase in root thickness ± SD of the four groups among the fi ve evaluation periods.

Group IV (DAP-3) Mean
In our study, Saline drops were added and mixed with the antibiotic combination until a creamy paste was achieved.
This is consistent with most case reports, in which there was no attempt to deliberately deliver a specifi c concentration of the drugs. Instead, drugs were mixed until a certain physical consistency was achieved that was deemed suitable by clinicians [4].
Propolis was used as an intra-canal irrigant and has been found to be effective in reducing endotoxins and inhibiting E.
faecalis, Streptococcus aureus, Candida albicans and E. coli [22]. Also, the co-adjuvant activity of propolis in antimicrobial therapy was confi rmed [23]. In vitro experiments have shown that propolis presents a high level of artepillin-C, inhibiting the cyclo-oxygenase pathway and eicosanoid synthesis [24]. In addition, studies have observed that propolis promotes regeneration of dental pulp [25].
Immature permanent teeth with necrotic pulp and an apical opening that measures 1 mm or larger were selected because they are considered suitable candidates for treatment [26,27]. Mechanical instrumentation is contraindicated, not only to avoid further weakening the thin root canal walls, but also to protect the vitality of stem cells of apical tissues [2]. However, Lin et al. 2014 published a failed clinical case of regenerative endodontic therapy due to the bacterial biofi lms that remained fi rmly attached to the apical canal walls because   of the lack of mechanical debridement [28]. Therefore, there is a trend for even slightly mechanical instrumentation in recent publications [4,29], which was made in our study.
Our clinical observations are in agreement with Paryani and Kim [30]. Our clinical results are also in full agreement with a retrospective study, which demonstrated 100% survival rate of teeth after regenerative treatment [31]. In a retrospective study of 30 cases, followed for up to 1 year, only two cases had complications, which were restricted to discomfort or discoloration [17], which is consistent with our study.
The bone density results in our study showed an increase in the radio-opacity of previously rarefacted areas. This indicates a favorable healing response to the treatment. In the majority of our cases, improvement or resolution of the apical lesion was evident in approximately 6 months. Root elongation and apical closure, with thickening of the root canal walls, was evident after 12-16 months postoperatively.
Our fi ndings concerning the increase in length and thickness were in agreement with many authors [5,19,26,32].
Our fi ndings were also in agreement with other authors [33,34], who reported an increase in length and thickness without quantitative measurements. The results of our study are in disagreement with Lin et al., [28], where the failure was attributed to inadequate root canal disinfection without mechanical removal of biofi lm and bacteria in dentinal tubules.

Conclusions
All tested intracanal medicaments paved the way for successful pulp revascularization process in immature permanent anterior teeth, which was evident by continued root growth and resolution of periapical radiolucency. Thereofore, the null hypothesis was accepted.

Recommendations
Follow up visits should be scheduled every 6 months, rather than 3 months, to observe root changes after pulp